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6-His-MBP-TEV-FnCpf1 vector

Basic Vector Information

Gene/Insert name FnCpf1 (humanized) Alt name type V CRISPR-associated protein Cpf1 (humanized) Alt name Cpf1 (humanized) Alt name Francisella tularensis subsp. novicida U112 Cpf1 (humanized) Species Francisella tularensis subsp. novicida U112 Insert Size (bp) 3910 Promoter T7 Tags / Fusion Proteins 6xHis (N terminal on backbone) MBP (N terminal on insert) TEV site (N terminal on insert) Nucleoplasmin NLS (C terminal on insert) 3xHA (C terminal on insert)

Basic Vector Information
Vector Name 6-His-MBP-TEV-FnCpf1 Antibiotic Resistance Ampicillin
Length 10685 bp Type CRISPR Plasmids
Source Feng Zhang Copy Number High copy number
Promoter T7 Cloning Method Enzyme Cut
5' Primer T7 promoter 3' Primer T7 terminator
Fusion Tag MBP Expression Method IPTG induced

6-His-MBP-TEV-FnCpf1 vector Vector Map

6-His-MBP-TEV-FnCpf1 vector Vector Map

6-His-MBP-TEV-FnCpf1 vector Sequence

LOCUS       Exported               10685 bp ds-DNA     circular SYN 04-JUL-2018
DEFINITION  Bacterial expression plasmid for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    6-His-MBP-TEV-FnCpf1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10685)
  AUTHORS   Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, 
            Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin 
            EV, Zhang F
  TITLE     Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas 
            System.
  JOURNAL   Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 
            10.1016/j.cell.2015.09.038.
  PUBMED    26422227
REFERENCE   2  (bases 1 to 10685)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, July 4, 2018 from SnapGene 3.2.1
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10685
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      532..1120
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    1306..1445
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(1547..1738)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2547..3629)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3630..3707)
                     /gene="lacI"
                     /note="lacI promoter"
     promoter        4016..4034
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4035..4059
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             4074..4096
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4104..9326
                     /codon_start=1
                     /note="6His-MBP-TEV-FnCpf1-NLS-3xHA"
                     /translation="MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTV
                     EHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDA
                     VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY
                     FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAE
                     AAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNK
                     ELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPN
                     IPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGASTGSLVPRGSASENLYFQGMSIY
                     QEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIE
                     EILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNL
                     FNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKG
                     FHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE
                     ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKG
                     INEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQ
                     IAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAV
                     LEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFE
                     EILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNN
                     LLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDE
                     KFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEG
                     YKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEF
                     NIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISE
                     SYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAEL
                     FYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFK
                     SSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDR
                     MKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFED
                     LNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKK
                     MGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFE
                     FSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIE
                     YGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDS
                     RQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNKR
                     PAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             4110..4127
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     regulatory      5082..5091
                     /regulatory_class="other"
                     /note="Kozak sequence"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             5244..5261
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             5268..5288
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /note="TEV site"
                     /translation="ENLYFQG"
     CDS             9189..9236
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9243..9323
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /note="3xHA"
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             9348..9371
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /note="FLAG"
                     /translation="DYKDDDDK"
     CDS             9372..9416
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /note="AviTag(TM)"
                     /translation="GLNDIFEAQKIEWHE"
     CDS             9432..9449
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     terminator      9516..9563
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      9600..10055
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        10081..10185
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             10186..361
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"

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